| Abstract: | Background: EpCAM is a type I transmembrane glycoprotein with an extracellular domain (EpEX) and an intracellular domain (EpICD), which have 265 and 26 amino acid residues, respectively. EpCAM is highly expressed in advanced epithelial cancers and tumor-initiating cells, but its role in cancer progression remains to be elucidated. Methods: To identify cell signaling pathways that are stimulated by EpEX, we used a Human Phospho-RTK Array Kit to screen for phosphorylation of RTKs. Inhibition of EpEX-induced EGFR-PI3K-AKT signaling was analyzed by small molecule inhibitors or shRNA knockdown. Results: Here, we found that EpEX activated AKT signaling, thereby inducing the phosphorylation and nuclear exclusion of FOXO3a. The EGFR inhibitor, AG1478, and MEK inhibitor, U0126, both decreased the production of EpICD, which was found to be necessary for nuclear accumulation of β-catenin protein and expression of HIF1α target genes in vitro and in mouse xenograft models. We also demonstrated that treatment with an anti-EpCAM neutralizing antibody, EpAb2-6, decreased the ADAM17 and γ-secretase activity, the EpCAM-downstream gene expression, and tumor colony and sphere formation. We also found that EpAb2-6 inhibited EpEX-activated EGFR-PI3K-AKT signaling and induced apoptotic signaling through FOXO3a activation of HTRA2 gene expression. Importantly, we also showed that EpAb2-6 inhibited the nuclear translocation of EpICD and oncogenic signaling through β-catenin. Finally, in both metastatic and orthotopic animal models of colorectal cancer, EpAb2-6 therapy exhibited an antitumor effect and markedly extended the survival time of mice. Conclusions: Our results demonstrate that EpEX contributes to malignancy by functioning as a growth factor, which activates EpICD-mediated signaling, thereby enhancing colon cancer cell survival. To the best of our knowledge, hEpAb2-6 is the first humanized anti-EpCAM antibody to directly trigger apoptosis in cancer cells. Thus, we provide novel insight into EpEX-EGFR signaling, which can be considered as a promising target for treatment of colon cancer. |
